Culturing of Ventricle Cells at High Density and Construction of Engineered Cardiac Cell Sheets Without Scaffold

Abstract

SummaryIn natural heart tissue, cell density is about 1.0 × 10 8 /cm 3 , and the cell metabolism is very active. Therefore, culturing heart cells in 3-dimensions at high density and construction of engineered cardiac tissue in vitro is very difficult. The aim of this study was to simulate 3-dimensional culturing of cardiac cells and pursue a novel method to construct engineered cardiac tissue in vitro. The isolated neonatal rat ventricle cells were cultured at a high seeding density of 1 × 10 6 /cm 2 . The cells at high density metabolized actively;the glucose consumption and lactic acid production of ventricle cells were much greater than those of fibroblasts cultured at the same density. The pH value of the culture medium of ventricle cells consistently decreased more rapidly. These cultured ventricle cells contained vascular endothelial cells, cardiomyocytes, and smooth muscle cells that appeared close to each other, and had overlapping nuclei and plenty of extracellular collagen. The cells at high density were treated with 0.2% trypsin to construct engineered cardiac cell sheets without scaffold. The engineered cardiac cell sheets could beat and roll up spontaneously, each sheet was 3 to 5 cells thick, and contained abundant cardiomyocytes and extracellular collagen.In conclusion, cells cultured at high-density in vitro grew well in a 2-dimensional culturing environment, formed "quasi 3-dimension" culturing, and engineered cardiac cell sheets comprised of several layers of cells were constructed. This study provides some guidance for cardiac tissue engineering and a novel method to construct engineered cardiac tissue without scaffold. (Int Heart J 2009; 50: 653-662)

Extracellular Matrix of Mechanically Stretched Cardiac Fibroblasts Improves Viability and Metabolic Activity of Ventricular Cells

“…In this study, the in vitro cultured ventricular cells were composed of cardiomyocytes, fibroblasts, vascular endothelial cells, smooth muscle cells and other cell types 15, 29, which is consistent with normal cardiac tissue. Therefore, the evaluation of metabolism and function of cultured ventricular cells should correspond to that of intact cardiac tissue.…”

“…The ventricular cells were isolated according to our previous method 15. The ventricles of rat hearts were minced, rinsed with PBS consisting of 1% penicillin streptomycin (Invitrogen), and treated with four times of 6-minute digestion in 0.25% trypsin solution (Invitrogen) and one time 20-minute digestion in 0.1% collagenase type I (Invitrogen) solution at 37 °C on a shaker (140 r/min).…”

Cardiac Fibroblast-Derived Extracellular Matrix Produced <I>In Vitro</I> Stimulates Growth and Metabolism of Cultured Ventricular Cells

“…The isolated ventricular cells were composed of cardiomyocytes, fi broblasts, vascular endothelial cells, smooth muscle cells, and other cell types, 6,17,32) which is consistent with normal cardiac tissue. Therefore, the values of cell metabolism and function that we measured in culture should correspond to values of metabolism and function of intact cardiac tissue.…”

Contractile force generation by 3D hiPSC-derived cardiac tissues is enhanced by rapid establishment of cellular interconnection in matrix with muscle-mimicking stiffness

“…To engineer physiologically relevant 3D cardiac tissue, choices of cell source and the scaffolding materials are of great importance. Given the cardiac tissue is characterized by high cell concentration (10 8 – 10 9 cells/mL)[11], reliable source of CMs is critical. Human induced pluripotent stem cells (hiPSCs) have emerged as an attractive source of human CMs since they can self-renew indefinitely and be directed towards CM differentiation efficiently (80 – 90%).…”